D-2-Hydroxy-4-Methylvalerate Dehydrogenase from Lactobacillus Delbrueckii Subsp. Bulgaricus- I. Kinetic Mechanism and pH Dependence of Kinetic Parameters, Coenzyme Binding and Substrate Inhibition (original) (raw)

The steady-state kinetics of D-2-hydroxy-4-methylvalerate dehydrogenase have been studied at pH 8.0 by initial velocity, product inhibition, and dead-end inhibition techniques. The mechanism is rapid-equilibrium ordered in the NAD' plus D-2-hydroxy-4-methylvalerate direction, and steady-state ordered in the other direction. In both cases coenzyme is the first substrate added and both the E-NADH-~-Zhydroxy-4-methylvalerate and E-NAD' -2-0x0-4-methylvalerate give rise to abortive complexes which cause excess substrate inhibition. Steady-state measurements show that the rate-limiting step in both directions at pH 8.0 is between formation of the enzyme-coenzyme-substrate ternary complex and the release of the first product of the reaction. Transient kinetics combined with primary kinetic deuterium isotope effects show that in the NADH -NAD' direction there is a slow, rate-limiting rearrangement of the E-NADHoxoacid complex while hydride transfer is very fast. The release of NAD' at pH 8.0 is 200-times faster than k,,, (NADH -NAD') whereas the release of NADH is only 5-times faster than k,,, (NAD' -NADH). The pH dependence of NADH binding depends upon the presence of two ionizable residues with a pK, of about 5.9. The pH dependence of kinetic parameters is explained by a third ionizable residue with pK, values 7.2 (in the E-NADH complex) and c6.4 (in the E-NAD' complex) which may be the proton donor and acceptor for the chemical reaction.